| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
Correspondence
Soon Dong Lee
sdlee{at}cheju.ac.kr
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
A transmission electron micrograph of a cell of strain HST3-14T is available as a supplementary figure with the online version of this paper.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
, to accommodate a strain (previously misclassified as ‘Pseudomonas riboflavina’) that comprised Gram-negative, obligately aerobic, motile rods and was phylogenetically related to members of the class Alphaproteobacteria. The genus currently contains five species with validly published names: Devosia riboflavina (Foster, 1944; Nakagawa et al., 1996) as the type species of the genus, Devosia neptuniae (Rivas et al., 2003), Devosia limi (Vanparys et al., 2005), Devosia soli (Yoo et al., 2006) and Devosia insulae (Yoon et al., 2007), recovered from soils, a plant and a commercial nitrifying inoculum. D. neptuniae is known as a non-rhizobial bacterium capable of forming nitrogen-fixing root-nodule symbioses with leguminous plants (Rivas et al., 2002). Three uncultured Devosia strains, with the provisional name ‘Candidatus Devosia euplotis’, were identified from among endosymbionts of marine ciliated protozoa, based on 16S rRNA gene sequence analysis (Vannini et al., 2004). According to a previously emended description (Rivas et al., 2003), the genus belonged phylogenetically to the family Hyphomicrobiaceae in the order Rhizobiales. A novel marine bacterium, comprising Gram-negative, aerobic, chemoheterotrophic, motile, short rods, was isolated from beach sediment in Jeju, Republic of Korea. An analysis of the 16S rRNA gene sequence revealed that the isolate belonged to the order Rhizobiales and was related to members of the genus Devosia. In this paper, the classification of strain HST3-14T is described by means of a polyphasic approach based on physiological, chemotaxonomic and phylogenetic analyses.
Sediment samples were taken at a depth of 1.5 m below the surface, placed into sterilized 50 ml Falcon tubes and stored at 4 °C. Sediment samples (1 g) were air-dried for 24 h under laminar flow and stamped directly onto plates of SC-SW agar (Lee, 2006) supplemented with 60 % (v/v) natural seawater, using a sterile rubber stopper. Following incubation at 30 °C for 7 days, the colony on the isolation plate was streaked on marine agar 2216 (MA; Difco). Pure cultures were maintained at –20 °C and –80 °C as 20 % glycerol suspensions supplemented with 60 % (v/v) natural seawater.
To test the requirement of strain HST3-14T for natural seawater or artificial sea salts (Sigma) for growth, the strain was grown on yeast extract-malt extract agar (Shirling & Gottlieb, 1966), trypticase soy agar (Difco) and nutrient agar with/without supplementation with 60 % (v/v) natural seawater and SS agar [3.1 % sea salts (Sigma), 0.15 % Bacto soytone (Difco), 0.45 % Bacto tryptone (Difco) and 1.7 % agar, pH 7.3]. Strain HST3-14T showed a requirement for seawater or sea salts (Sigma) for growth and did not grow on yeast extract-malt extract agar, trypticase soy agar or nutrient agar without the addition of seawater.
DNA extraction and PCR amplification and sequencing of the 16S rRNA gene were performed as described elsewhere (Lee, 2006): an almost-complete 16S rRNA gene sequence (1406 nt) was determined for strain HST3-14T. A preliminary BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) revealed that the isolate was related to members of the genus Devosia in the order Rhizobiales. The CLUSTAL_X program (Thompson et al., 1997) was used to align the sequence with corresponding sequences from Devosia species and related taxa retrieved from public databases. Phylogenetic analyses were carried out using several programs contained in the PHYLIP package (Felsenstein, 1993). A distance-based tree was developed using the method of Jukes & Cantor (1969) and the neighbour-joining treeing algorithm (Saitou & Nei, 1987) and the topology of the tree was evaluated using bootstrap analysis (Felsenstein, 1985).
In a neighbour-joining tree (Fig. 1) based on 1356 unambiguous nucleotides present in the 16S rRNA gene sequences of all strains, strain HST3-14T formed a distinct sub-branch within the genus Devosia. The affiliation of strain HST3-14T to the genus Devosia was supported by a high bootstrap value (100 %) and was also found in trees constructed using the maximum-parsimony and maximum-likelihood methods. The 16S rRNA gene sequence similarities between strain HST3-14T and members of the genus Devosia were as follows: 96.8 % to D. riboflavina DSM 7230T, 96.7 % to D. neptuniae J1T, 96.5 % to D. soli GH2-10T, 96.2 % to D. limi LMG 22951T, 96.2 % to ‘Candidatus Devosia euplotis’, 96.2 % to ‘D. terrae’ DCY11, 95.4 % to D. insulae DS-56T and 95.2 % to ‘Devosia ginsengisoli’ Gsoil 326. In general, organisms sharing less than 97.0 % 16S rRNA gene sequence similarity do not have DNA–DNA reassociation value greater than 70 % (a cut-off point representing a criterion for delineating a genomic species; Stackebrandt & Goebel, 1994), and DNA–DNA hybridization was therefore not performed.
|
9c, C18 : 112t and C18 : 17c; 48.6 %), C18 : 0 (17.5 %), C16 : 0 (15.9 %), 10-methyl C19 : 0 (4.1 %), C12 : 0 (3.8 %), cyclo-C17 : 0 (1.4 %), a mixture of C16 : 17c and/or iso-C15 : 0 2-OH (2.0 %) and an unknown acid with an equivalent chain-length of 18.856 (4.1 %). This major fatty acid profile is typical of members of the class Alphaproteobacteria, but differs from those of members of the genus Devosia (Vanparys et al., 2005; Yoo et al., 2006; Yoon et al., 2007) in that it lacks 10-methyl C18 : 17c. A comparative analysis of the fatty acids of the isolate and members of the genus Devosia (of terrestrial origin) under the same conditions was not performed because strain HST3-14T has a requirement for seawater or artificial sea salts for growth.
An isoprenoid quinone analysis was performed using HPLC as described previously (Kroppenstedt, 1985), with Q-10 (Sigma) as the standard ubiquinone. The G+C content of the DNA was determined using HPLC (Mesbah et al., 1989). Cell biomass was obtained from cultures grown in marine broth (Difco) at 30 °C. The major ubiquinone in strain HST3-14T was Q-10. Q-11, which is the major isoprenoid quinone in D. insulae, was not detected. The DNA G+C content of strain HST3-14T was 59.1 mol%. The presence of nodD and nifH genes (encoding components of legume nodulation and symbiotic nitrogen fixation, respectively) was examined as described previously (Rivas et al., 2002), using D. neptuniae LMG 21357T as the positive control and D. soli KACC 11509T as the negative control. Plasmid isolation was performed using the Wizard Plus SV Minipreps DNA purification system (Promega). nodD and nifH genes were amplified by PCRs from both total DNA and plasmid DNA of D. neptuniae LMG 21357T, but not from DNA of strain HST3-14T or D. soli KACC 11509T.
The morphology and motility of strain HST3-14T were determined using phase-contrast microscopy and transmission electron microscopy on cells in the exponential phase of growth. The presence of flagella was assessed with a transmission electron microscope (JEM-1010; JEOL). The ability to degrade casein, starch, Tween 80, CM-cellulose, chitin and DL-tyrosine was determined using MA as the basal medium, as described previously (Gordon et al., 1974). DNA hydrolysis was investigated on DNase test agar (Difco) supplemented with 60 % (v/v) natural seawater. Growth on MA was tested at 4, 10, 20, 30, 37 and 42 °C and at pH 4.1–12.1. Catalase activity was determined with a 3 % (v/v) hydrogen peroxide solution. Oxidase activity and the Gram stain were assessed using the method of MacFaddin (1980). Tolerance of NaCl and sea salts (Sigma) for growth was tested on MA at 1–7 % and 0.5–9 %, respectively. Utilization of carbon sources and activities of enzymes were examined using API 20NE and API ZYM kits according to the instructions of the manufacturer (bioMérieux). Cells of strain HST3-14T were grown on MA for 2 days at 30 °C and suspended in sea salts (Sigma) solution (2 %, w/v) before inoculation. The results of physiological and biochemical tests are given in the species description and in Table 1. Colonial morphology was observed after 7 days cultivation at 30 °C on MA: the colonies were small, smooth, circular, convex and light yellow to light brown in colour. The cells were strictly aerobic, non-spore-forming, Gram-negative rods and were motile by means of single, polar, monotrichous flagella (see Supplementary Fig. S1 available in IJSEM Online).
|
. On the basis of the phenotypic and phylogenetic data presented here, strain HST3-14T represents a novel species of the genus Devosia, for which the name Devosia subaequoris sp. nov. is proposed.
Description of Devosia subaequoris sp. nov.
Devosia subaequoris (sub.ae.quo'ris. L. prep. sub under; L. gen. n. aequoris of the sea; N.L. gen. n. subaequoris from under the sea, referring to the place where the type strain was isolated).
Cells are Gram-negative, non-spore-forming, oxidase-positive, catalase-positive, motile rods (0.7 µm wide and 1.2 µm long). On MA, colonies are very small (0.7–1.0 mm in diameter), circular, smooth, convex and light yellow to light brown in colour. Growth occurs between 20 and 42 °C, but not at or below 10 °C. The pH range for growth is 5.1–12.1. Seawater or sea salts is required for growth. Growth occurs on MA supplemented with 0–3 % NaCl or 0–3 % sea salts. Casein, starch, Tween 80, cellulose, chitin and DNA are not hydrolysed. DL-Tyrosine is not decomposed. In the API 20NE test, the result for aesculin degradation is positive. Assimilation of D-mannose, D-mannitol and maltose is weakly positive. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, gelatin hydrolysis and urease. D-Glucose, L-arabinose, N-acetylglucosamine, gluconate, caprate, adipate, malate, citrate and phenylacetate are not assimilated. In API ZYM tests, the results for alkaline phosphatase, esterase lipase (C8) and leucine arylamidase are positive. Weakly positive for esterase lipase (C4) and trypsin. Negative for lipase (C14), valine arylamidase, cystine arylamidase, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, 
-glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The predominant ubiquinone is Q-10. Major fatty acids are C18 : 1 (one or more of the isomers C18 : 19c, C18 : 112t and C18 : 17c; 48.6 %), C18 : 0 (17.5 %) and C16 : 0 (15.9 %). The G+C content of the DNA is 59.1 mol%.
The type strain, HST3-14T (=KCTC 12772T =JCM 14206T), was isolated from a sediment sample collected from Hwasun Beach in Jeju, Republic of Korea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[Abstract]
Foster, J. W. (1944). Microbiological aspects of riboflavin. J Bacteriol 47, 27–41.
Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24, 54–63.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics, pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
Lee, S. D. (2006). Kineococcus marinus sp. nov., isolated from marine sediment of the coast of Jeju, Korea. Int J Syst Evol Microbiol 56, 1279–1283.
MacFaddin, J. F. (1980). Biochemical Tests for Identification of Medical Bacteria, 2nd edn. Baltimore: Williams & Wilkins.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Nakagawa, Y., Sakane, T. & Yokota, A. (1996). Transfer of "Pseudomonas riboflavina" (Foster 1944), a Gram-negative, motile rod with long-chain 3-hydroxy fatty acids, to Devosia riboflavina gen. nov., sp. nov., nom. rev. Int J Syst Bacteriol 46, 16–22.
Rivas, R., Velázquez, E., Willems, A., Vizcaíno, N., Subba-Rao, N. S., Mateos, P. F., Gillis, M., Dazzo, F. B. & Martínez-Molina, E. (2002). A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) Druce. Appl Environ Microbiol 68, 5217–5222.
Rivas, R., Willems, A., Subba-Rao, N. S., Mateos, P. F., Dazzo, F. B., Kroppenstedt, R. M., Martínez-Molina, E., Gillis, M. & Velázquez, E. (2003). Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India. Syst Appl Microbiol 26, 47–53.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[Medline]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Vannini, C., Rosati, G., Verni, F. & Petroni, G. (2004). Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of ‘Candidatus Devosia euplotis’. Int J Syst Evol Microbiol 54, 1151–1156.
Vanparys, B., Heylen, K., Lebbe, L. & De Vos, P. (2005). Devosia limi sp. nov., isolated from a nitrifying inoculum. Int J Syst Evol Microbiol 55, 1997–2000.
Yoo, S.-H., Weon, H.-Y., Kim, B.-Y., Hong, S.-B., Kwon, S.-W., Cho, Y.-H., Go, S.-J. & Stackebrandt, E. (2006). Devosia soli sp. nov., isolated from greenhouse soil in Korea. Int J Syst Evol Microbiol 56, 2689–2692.
Yoon, J.-H., Kang, S.-J., Park, S. & Oh, T.-K. (2007). Devosia insulae sp. nov., isolated from soil, and emended description of the genus Devosia. Int J Syst Evol Microbiol 57, 1310–1314.
This article has been cited by other articles:
|
|
 |
|
  H.-Y. Xu, L.-P. Chen, S.-Z. Fu, H.-X. Fan, Y.-G. Zhou, S.-J. Liu, and Z.-P. Liu Zhangella mobilis gen. nov., sp. nov., a new member of the family Hyphomicrobiaceae isolated from coastal seawater Int J Syst Evol Microbiol, September 1, 2009; 59(9): 2297 - 2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Verma, M. Kumar, M. Dadhwal, J. Kaur, and R. Lal Devosia albogilva sp. nov. and Devosia crocina sp. nov., isolated from a hexachlorocyclohexane dump site Int J Syst Evol Microbiol, April 1, 2009; 59(4): 795 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Y. Hwang, K. D. Cho, W. Yih, and B. C. Cho Maritalea myrionectae gen. nov., sp. nov., isolated from a culture of the marine ciliate Myrionecta rubra Int J Syst Evol Microbiol, March 1, 2009; 59(3): 609 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Ryu, B. S. Chung, N. T. Le, H. H. Jang, P.-Y. Yun, W. Park, and C. O. Jeon Devosia geojensis sp. nov., isolated from diesel-contaminated soil in Korea Int J Syst Evol Microbiol, March 1, 2008; 58(3): 633 - 636. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
